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-MSH on food
intake, adiposity, c-Fos induction, and neuropeptide
expression
1 Program in Nutritional Sciences, Departments of 2 Psychiatry and Behavioral Sciences and 6 Medicine, University of Washington, Seattle 98195, 3 Puget Sound Veterans Affairs Health Care System, Seattle 98108, 7 Harborview Medical Center, Seattle, Washington 98104; 4 Department of Nutrition, University of California at Davis, Davis, California 95616; and 5 Department of Psychiatry, University of Cincinnati, Cincinnati, Ohio 45267
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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-Melanocyte-stimulating hormone (-MSH) is a hypothalamic
neuropeptide proposed to play a key role in energy homeostasis. To
investigate the behavioral, metabolic, and hypothalamic responses to
chronic central -MSH administration, -MSH was infused
continuously into the third cerebral ventricle of rats for 6 days.
Chronic -MSH infusion reduced cumulative food intake by 10.7%
(P < 0.05 vs. saline) and body weight by 4.3%
(P < 0.01 vs. saline), which in turn lowered plasma
insulin levels by 29.3% (P < 0.05 vs. saline). However, -MSH did not cause adipose-specific wasting nor did it
alter hypothalamic neuropeptide mRNA levels. Central -MSH infusion
acutely activated neurons in forebrain areas such as the hypothalamic
paraventricular nucleus, as measured by a 254% increase in c-Fos-like
immunoreactivity (P < 0.01 vs. saline), as well as
satiety pathways in the hindbrain. Our findings suggest that, although
an increase of central melanocortin receptor signaling acutely reduces
food intake and body weight, its anorectic potency wanes during chronic
infusion and causes only a modest decrease of body weight.
melanocortin; hypothalamus; body weight
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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-MELANOCYTE-STIMULATING
HORMONE (-MSH) and its analogs acutely suppress food intake
after intracerebroventricular administration in rats and mice
(6, 11, 14, 23,
29, 31, 40, 41). Several observations suggest a physiological role for this anorectic response to -MSH. For example, conditions associated with an increased drive to consume food, such as fasting (2,
5) and genetic leptin deficiency (26,
36), are accompanied by reduced expression of the gene
encoding proopiomelanocortin (POMC; the precursor molecule for -MSH)
in the hypothalamic arcuate nucleus (Arc). Conversely, POMC
mRNA is upregulated in the overfed state (15) and by acute
injections of leptin (26, 36). In addition,
rodents and humans with mutations of the gene encoding POMC, or POMC
processing enzymes, are obese and hyperphagic (20, 30).
After its release from axon terminals in hypothalamic areas
such as the paraventricular nucleus (PVN), -MSH binds to and activates neuronal melanocortin-4 (MC-4) receptors.
Intracerebroventricularly administered MC-4 receptor antagonists
acutely stimulate food intake (11, 33), and
MC-4 receptor-deficient mice (19) and humans
(42) have an obese phenotype, suggesting that this
receptor subtype plays a major role in the anorectic action of -MSH.
The agouti mouse is also obese, presumably due to ectopic
production the melanocortin receptor antagonist agouti in the brain
(11). Agouti-related peptide (AgRP) is a naturally
occurring endogenous antagonist to the MC-4 receptor that is
synthesized in a subset of Arc neurons adjacent to POMC cells.
Centrally administered AgRP blocks the catabolic action of -MSH and
increases food intake when given intracerebroventricularly, suggesting
that melanocortin signaling is essential to constrain food intake. AgRP
is colocalized with neuropeptide Y (NPY) in Arc neurons, and, similar
to NPY, AgRP is upregulated in response to fasting (16,
27), leptin deficiency (27, 43),
and streptozotocin-induced diabetes (18), suggesting that
AgRP also plays a physiological role in body weight regulation. We
therefore hypothesized that chronic intracerebroventricular infusion of
-MSH would promote a sustained state of negative energy balance,
leading to a reduction in body weight.
Candidate targets of first-order neurons releasing -MSH
include second-order neurons that express the catabolic peptide
corticotropin-releasing hormone (CRH), contained in the parvocellular
region of the PVN, as well as second-order neurons expressing the
orexigenic peptide melanin-concentrating hormone (MCH) in the lateral
hypothalamic area (LHA). In support of this model, both the PVN and LHA
are supplied by melanocortin-containing projections from the Arc
(8, 9) and express MC-4 receptor mRNA
(7a, 28). In addition, expression of CRH and
MCH is altered by changes in energy balance (5, 32). We hypothesized, therefore, that CRH and MCH neurons
are targets of -MSH signaling, whereas POMC and AgRP gene expression appear to be regulated by a direct action of leptin, binding to leptin
receptors located on Arc neurons transcribing these peptides (1, 7, 10). Thus we also
hypothesized that intracerebroventricular -MSH infusion would alter
CRH and MCH gene expression via melanocortin receptors located in areas
of the brain containing these neuropeptides, but would not alter POMC
or AgRP expression.
To test these hypotheses, we administered -MSH into the third
ventricle of rats over 6 days to ascertain its chronic effects on food
intake, body weight, plasma hormone levels, body composition, and
neuropeptide expression in the hypothalamus. In addition, we
hypothesized that a single injection of -MSH would acutely activate
neuronal substrates in energy regulation pathways. To identify these
sites, we injected rats in the third ventricle with -MSH and
quantified c-Fos-like immunoreactive nuclei in the forebrain and hindbrain.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals
All studies used male Long-Evans rats (300-350 g) from the breeding colony maintained by the Department of Psychology at the University of Washington housed individually in wire-mesh hanging cages (experiments 1 and 3) or male Wistar rats weighing 300-350 g (Simonsen Laboratories, Gilroy, CA) were housed in polycarbonate cages (experiment 2) in a temperature-controlled vivarium on a 12:12-h light-dark schedule. Unless otherwise specified, animals were given free access to pelleted rat chow (Harlan-Teklad, Madison, WI, experiments 1 and 3; Ralston Purina, St. Louis, MO, experiment 2) and water at all times. All procedures were performed according to institutional guidelines of the Animal Care and Use Committee at the Seattle Veterans Affairs Medical Center and University of Washington.Cannula Placement
Rats were habituated with daily handling for 1 wk before surgery. After anesthesia induced by intraperitoneal injection of ketamine-xylazine (60 mg/kg ketamine and 8 mg/kg xylazine), a 21-gauge cannula (Plastics One, Roanoke, VA) was placed stereotaxically into the third ventricle using a previously described method (37, 39). Cannula placement was verified 1 wk after surgery by intracerebroventricular injection of 10 ng angiotensin II (American Peptide, Sunnyvale, CA) diluted in 1 µl saline. Animals not consuming at least 5 ml water 30 min postinjection were excluded as cannulation failures (4% of all rats). Experiments were performed at least 2-3 wk after surgery.Experimental Protocols
Experiment 1: acute effect of -MSH on food intake.
Cannulas were surgically implanted in the third ventricle of Long-Evans
rats, as described above. Food hoppers were removed from the animal
cages at 1500 and weighed. At 1600, animals were injected in
the third ventricle with either 5 µl of sterile, 0.9% preservative-free saline (Fujisawa USA, Deerfield, IL) or an equal volume of human -MSH (Peninsula Laboratories, Belmont, CA) in saline
using an injector (Plastics One) attached by polyethylene tubing to a
25-µl glass syringe (Hamilton, Reno, NV). A study comparing doses of
0, 2.5, 25, and 50 µg -MSH (n = 5-7/group) was performed. For each trial, infusate was delivered manually over a
period of 2 min, after which time animals received an intramuscular injection of 8 mg gentamicin sulfate to prevent infection and were
immediately returned to their home cages. Food hoppers were replaced on
the cages at 1730, and lights out occurred at 1800. Food-intake data
were collected at the 1-, 2-, 3-, 4-, 16-, and 24-h time points. A
recovery period of at least 6 days was allowed between trials.
Experiment 2: chronic intracerebroventricular -MSH infusion.
On the basis of preliminary results showing that a single
intracerebroventricular injection of 10 µg -MSH reduces 4-h food intake in Wistar rats and that a chronic intracerebroventricular infusion of 96 µg/day is no more effective than 24 µg/day at
reducing food intake and body weight after 6 days (data not shown), we selected a dose of 24 µg/day for this study. Three weeks after cannula placement, each rat was anesthetized by intraperitoneal injection of ketamine-xylazine and received a subcutaneous osmotic minipump (Azlet model 2001, Palo Alto, CA) delivering either 1 µg/µl human -MSH in 0.9% saline vehicle at a rate of 1 µl/h
(24 µg/day; n = 9) or vehicle alone
(n = 7) via a polyethylene catheter that was connected
to the ventricular cannula. A saline-infused group pair-fed to the food
intake of the -MSH group (n = 9) was also included
to determine if some responses to intracerebroventricular -MSH were
secondary to reduced food intake. The amount of chow provided to each
pair-fed animal on each treatment day was equal to the measured amount
of chow consumed by its -MSH-treated partner during the previous
24-h period. Animals received continuous intracerebroventricular infusions of either -MSH or saline for 6 days and were lightly anesthetized by brief exposure to carbon dioxide before decapitation between 1100 and 1300 on the 6th day of infusion. Trunk blood, carcasses, and brains were collected upon decapitation. Blood was
centrifuged, and plasma was stored at 
20°C and brains were stored
at 80°C. Carcasses were stored at 20°C.
Experiment 3: acute effect of -MSH on c-Fos-like
immunoreactivity in the brain.
Three weeks after cannula placement in the third ventricle, male
Long-Evans rats were assigned to one of two weight-matched groups and
injected in the third ventricle with either 5 µl of saline
(n = 5) or 20 µg -MSH in saline (n = 10). Injections were performed between 1000 and 2000, after which the
animals were immediately returned to their home cages and their food
hoppers were removed. After 110 min, rats were anesthetized with
pentobarbital sodium (60 mg ip) and transcardially perfused with
isotonic saline of neutral pH followed by a 4% paraformaldehyde
solution. Brains were removed immediately and postfixed in
paraformaldehyde for 1 wk before assay for c-Fos-like immunoreactivity (cFLI).
Assays and Data Analyses
Plasma assays. Radioimmunoassays were used to measure plasma levels of corticosterone and immunoreactive insulin as previously described (13, 37). Plasma glucose was determined by the glucose oxidase method (Beckman Instruments, Brea, CA). Plasma leptin levels were determined by radioimmunoassay using a rat-specific antibody (Linco, St. Louis, MO) (21).
Analysis of body composition. Postmortem body fat and nonfat mass were measured by dual-energy X-ray absorptiometry (DEXA; QDR 1500, Hologic, Waltham, MA) using software designed for rats. The DEXA scanner was calibrated to quantify nonfat mass as lean tissue comprised of 60% water, excluding bone mass. A tissue calibration scan was performed every 4 h to correct for DEXA performance variables over the course of the analysis. Intra-assay coefficient of variation for fat mass was 6.3%. Validation of DEXA for analysis of body fat stores in rats has been documented previously (3, 24, 44).
In situ hybridization to AgRP, POMC, CRH, and MCH mRNA. Brains from experiment 2 were immediately frozen on crushed dry ice and subsequently sectioned coronally at 14 µm in a cryostat and mounted on RNAse-free slides. Riboprobes complementary to rat POMC (a generous gift of Dr. Robert Steiner), MCH, and AgRP mRNAs (constructs provided by Dr. Tina M. Hahn) were used for hybridization after labeling with 33P, as previously described (36). Hybridization for CRH mRNA was performed using a 33P-labeled antisense oligonucleotide probe based on cDNA sequences of rat CRH genes, as described elsewhere (39). Slides for in situ hybridization (ISH) to POMC mRNA were selected from the region of the Arc rostral to the ventromedial hypothalamic nucleus (VMN), and sections for MCH and AgRP were taken from the midregion of the Arc, at the level of the VMN. Slides for CRH mRNA were selected from the PVN. All slides were selected by an investigator blinded to the treatment group. Labeled slides were washed under high-stringency conditions and opposed to X-ray film to generate autoradiographs, which were analyzed by computer densitometry. With the use of a standard curve, autoradiographic optical density and hybridization area were determined on six to eight sections per rat using the MCID computer densitometry system (Imaging Research, St. Catherine's, Ontario, Canada). The product of hybridization area (pixels) and density (µCi/pixel) was used as an index of overall neuropeptide mRNA levels, which are expressed as percentage of mean control values (34-36, 39).
cFLI. Each postfixed brain was rinsed two to three times in PBS and sectioned at 50 µm on a Vibratome in a PBS bath. Coronal sections taken from the forebrain and horizontal slices from the hindbrain were processed for cFLI as described in detail elsewhere (40). Sections were mounted on slides, and the number of cFLI-positive cell nuclei was quantified in specific brain areas using the MCID computer grain counting system (Imaging Research).
Statistical Analyses
All statistical analyses were carried out using Prism 2.01 (GraphPad Software, San Diego, CA) statistical software. Data are presented as group mean values (±SE). For experiments with greater than two study groups, comparisons were performed with one-way ANOVA and Newman-Keuls post hoc test. A Student's t-test was used for two-group comparisons. A P value
0.05 between group mean values was considered statistically significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Experiment 1: Acute Effect of Intracerebroventricular -MSH
Infusion on Food Intake
-MSH 2 h
before the dark cycle caused a nonsignificant 27.5% decrease of
cumulative 4-h food intake in nonfasted rats (Fig.
1). Food intake was significantly suppressed by 42.1% (7.9 ± 0.5 vs. 4.6 ± 0.8 g,
P < 0.05) after 25 µg intracerebroventricular
-MSH, and no additional food intake suppression was detected at the
50-µg dose. Twenty-four hours after -MSH infusion, cumulative food
intake and body weight across all dose groups did not differ
significantly from vehicle-treated control values.
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Experiment 2: Chronic Intracerebroventricular -MSH Infusion
Food intake.
Cumulative food intake over 6 days was reduced by 10.7% (119.3 ± 5.0 vs. 106.5 ± 3.9 g, P < 0.05) in rats
receiving 24 µg/day -MSH as a continuous intracerebroventricular
infusion compared with saline-treated controls fed ad libitum (Table
1). Analysis of the time
course of this effect demonstrated a 39.9% inhibition of food intake
that occurred during the first 24 h (12.7 ± 2.3 vs. 7.6 ± 0.9 g, P < 0.05), with food intake returning
to near normal levels on subsequent days. After an initial suppression of energy intake, therefore, the anorectic effect of -MSH was no
longer detected. By design, the intake of the saline-treated, pair-fed
group was matched to that of the -MSH group and was also
significantly below that of controls fed ad libitum.
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Body weight and composition.
As summarized in Table 2, mean body
weight at baseline was similar among the three groups of rats. When
expressed as a percentage of initial body weight, weight at the time of
death was decreased by 3.9 ± 0.8% in -MSH-treated rats and
3.1 ± 0.9% in the saline-infused pair-fed animals
(P < 0.01 for both comparisons) and remained unchanged
in the saline-infused control group. When expressed as a percentage of
final body weight of the saline control group, weight was decreased by
4.3% in the -MSH group and 3.6% in the pair-fed group. Final body
weight was not significantly different between the saline-infused
pair-fed and the -MSH-infused groups.
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-MSH-infused animals and
0.9 g less in the pair-fed group [P = not
significant (NS) between all groups], whereas lean mass was reduced by
15.7 and 13.7 g in the -MSH and pair-fed groups, respectively
(P = NS between all groups, Table 2). Total weight loss
was, therefore, comprised of decreases in both fat and lean mass, and
percent body fat was not significantly different in either the
-MSH-infused or pair-fed groups compared with the ad libitum-fed
controls (0.3% in the -MSH-infused group and +0.3% in the
pair-fed group).
Plasma values.
Nonfasting plasma insulin levels were reduced by 29.3% in the -MSH
group vs. saline-treated rats fed ad libitum (207.3 ± 25.7 vs.
293.1 ± 23.9 pmol/l, P < 0.05) and by 38.5% in
the pair-fed group vs. saline (180.3 ± 11.1 pmol/l,
P < 0.01 vs. saline-infused rats fed ad libitum), but
the -MSH and pair-fed groups were not significantly different (Fig.
2A). Plasma glucose levels
were not significantly different among groups (Fig. 2A).
Plasma leptin was decreased nonsignificantly by 20.1% in the -MSH
group compared with the saline group (1.2 ± 0.1 vs. 1.5 ± 0.2 ng/ml) and was decreased by 34.4% in the saline pair-fed group
compared with controls fed ad libitum (1.0 ± 0.1 ng/ml,
P < 0.05)(Fig. 2B). Plasma corticosterone
levels were increased by 51.0% in the -MSH-infused group compared
with the saline-infused group (94.1 ± 33.13 vs. 62.3 ± 15.5 ng/ml) and by 7.2% in the saline pair-fed group (66.8 ± 14.22 ng/ml) (Fig. 2B), although neither difference reached statistical significance.
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ISH.
As expected, hybridization to both POMC and AgRP mRNA in the
hypothalamus was detected only in the Arc. POMC mRNA levels in the
rostral Arc were nonsignificantly suppressed by 13.1 ± 4.0 and
16.1 ± 9.7% in the -MSH and pair-fed groups relative to
saline controls fed ad libitum (Fig. 3).
By comparison, AgRP mRNA levels in the Arc were elevated by 30.4 ± 10.1 and 12.6 ± 8.6% in the -MSH and pair-fed groups,
respectively, compared with ad libitum-fed controls, although neither
difference reached statistical significance. CRH mRNA levels in the PVN
were decreased by 18.2 ± 10.4% and increased by 24.2 ± 38.9% in the -MSH and pair-fed groups, respectively, compared with
saline controls (P = NS for both comparisons). MCH mRNA
levels in the LHA were increased by 121.1 ± 51.3 and 111.3 ± 41.0% in the -MSH and pair-fed groups compared with saline controls (P = NS for both comparisons).
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Experiment 3: Acute Effect of -MSH on cFLI in the Brain
-MSH at a dose of 20 µg (7.4 ± 1.9 vs. 53.8 ± 7.0 cFLI-positive nuclei,
P < 0.001) and was nonsignificantly increased by 185%
in the NTS. In the forebrain, -MSH also increased fos
staining in the supraoptic nucleus (SON), the PVN, the central nucleus
of the amygdala (CeA), and in the Arc. The number of nuclei with cFLI
in the SON was increased with intracerebroventricular -MSH by
1,142% (7.2 ± 1.9 vs. 89.4 ± 17.0 cFLI-positive nuclei,
P < 0.01), in the PVN by 254% (67.3 ± 32.2 vs.
238.0 ± 27.9 cFLI-positive nuclei, P < 0.01),
and in the CeA by 204% (19.0 ± 7.6 vs. 57.7 ± 10.4 cFLI-positive nuclei, P < 0.05). The number of
cFLI-positive nuclei in the Arc was nonsignificantly increased by 46%
(Fig. 4).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Summary of Findings
The melanocortin system is proposed to be an important hypothalamic pathway involved in energy homeostasis (25, 38). To compare the response to acute and chronic central melanocortin receptor stimulation and to investigate the effect of increased central nervous system (CNS) melanocortin signaling on hypothalamic and brain stem areas associated with energy homeostasis, we infused-MSH into the third cerebral ventricle of
adult male rats. We found that a single intracerebroventricular
injection of -MSH, at a dose that reduces 4-h intake, activated
neurons (as indicated by increased cFLI in cell nuclei) in several
forebrain areas, as well as in the lPBN, an important metabolic relay
area of the brain stem. A reduction in body weight, however, was not
observed after a single intracerebroventricular -MSH injection. In
contrast, continuous intracerebroventricular administration of -MSH
for 6 days did result in sustained decreases of both food intake and body weight, although the anorectic response to chronic infusion of
-MSH was greatest during the first 24 h of infusion and was not
significant thereafter. Moreover, a chronic infusion of -MSH did not
selectively reduce body fat and failed to alter expression of specific
hypothalamic neuropeptide mRNAs. Therefore, although -MSH acts in
the CNS to induce a state of negative energy balance, its efficacy
wanes with chronic administration, suggesting that a competing
mechanism attenuates its effectiveness.
Acute Injection of -MSH
-MSH on food intake, we measured food consumption in response to
different doses of -MSH administered to conscious, nonfasted rats
2 h before the beginning of the nocturnal cycle. We found that,
although a 25-µg injection of -MSH reduced food intake by 42.1%
over 4 h compared with saline-injected controls (P < 0.05, experiment 1), a compensatory increase of food
intake occurred subsequently, such that neither 24-h food intake nor
body weight was significantly affected. Higher doses were no more
effective in their ability to suppress food intake or to induce weight loss.
This finding differs from the effects of the synthetic
melanocortin receptor agonist MTII, which, at a dose of 1 nmol
icv, elicits a more sustained anorexia and a significant
weight loss after 24 h (40) despite an initial
response (at 4 h) comparable to that of 25 µg (15 nmol) of
-MSH. A 10-µg icv (6 nmol) dose of Nle4DPhe7alpha
(NDP-MSH), an -MSH analog, also maintains a significant
34% suppression of food intake over a 24-h period compared with
vehicle control (6). Because MTII and NDP-MSH are
engineered to bind the MC-4 receptor with higher affinity and, as
synthetic compounds, may be cleared less readily than -MSH
(17), the prolonged effects of MTII and NDP-MSH on food intake could reflect a sustained increase of neuronal melanocortin signaling. It is therefore possible that the short duration of -MSH-induced anorexia compared with MTII and NDP-MSH is due simply to a relatively short-lived stimulation of central melanocortin receptors, in which case, a chronic intracerebroventricular infusion of
-MSH should elicit a more durable anorexic response. To investigate this hypothesis, we measured the effect of continuous
intracerebroventricular administration of -MSH for 6 days.
Chronic Infusion of -MSH
-MSH-treated animals experienced
reduced food intake and body weight on the first day of infusion (presumably related to minipump implantation), animals infused with
-MSH consumed 39.9% less food than saline-infused, ad libitum-fed controls. Thus, whereas an acute intracerebroventricular injection of
25 µg -MSH suppressed food intake at 4 h, but not at 24 h, continuous infusion of a comparable dose (24 µg) induced a
cumulative reduction of food intake that was sustained for at least
24 h with no compensatory increase of food intake over the
subsequent 6 days of -MSH infusion. This demonstration that the
duration of -MSH-induced anorexia is increased by chronic
intracerebroventricular infusion supports the hypothesis that sustained
melanocortin signaling is necessary to achieve the more durable
anorexia induced by MTII or NDP-MSH. By day 2, however, food
intake differed by only 11.6% between these two groups and was only
5.9% lower for the remainder of the study. This attenuation of
-MSH-induced anorexia during continuous infusion is consistent with
the development of either melanocortin receptor downregulation,
compensatory responses involving other neuronal pathways, or both.
By day 6, the -MSH-infused group weighed 4.3% less than
the saline-infused controls, whereas saline-infused animals pair-fed to
the -MSH group exhibited a 3.6% weight loss that was not
significantly different from that seen in the -MSH-infused group.
Although these findings suggest that decreased food intake induced by
chronic infusion of -MSH is sufficient to explain the measured
weight reduction, they do not exclude an effect of -MSH on energy
expenditure. Postmortem body composition analysis indicated that the
percentages of lean and fat mass were similar between the -MSH and
the two control groups and that lost weight was comprised of both lean and fat tissue. Thus weight loss induced by chronic central
administration of -MSH is modest and is not comprised preferentially
of adipose tissue, as has been reported for leptin.
As expected, reduced body weight in both the -MSH and pair-fed
groups was associated with comparable reductions of plasma insulin and
leptin levels relative to ad libitum-fed controls. This finding
suggests that suppression of basal levels of these plasma hormones was
the consequence of reduced food intake and weight loss rather than an
independent effect of -MSH on hormone secretion. However, our study
did not investigate insulin secretory responses or systemic insulin
action. Thus further experiments are necessary to clarify this issue.
Measurements of plasma corticosterone and glucose levels also failed to
demonstrate independent effects of -MSH on these measures. Taken
together, these findings suggest that in normal rats, chronic
intracerebroventricular infusion of -MSH does not substantially
influence basal plasma levels of hormones involved in energy
homeostasis or glucose homeostasis independent of its effect to reduce
food intake and body weight.
Significant changes in hypothalamic neuropeptide mRNA levels were not
observed after chronic -MSH infusion. Although they did not reach
statistical significance, the direction and magnitude of changes of
POMC, CRH, MCH, and AgRP mRNA we detected are consistent with the
response predicted for the modest decrease of body weight and leptin
and insulin levels experienced by the -MSH and pair-fed groups.
Brain cFLI After -MSH Injection
-MSH was administered into the third cerebral ventricle, marked increases in cFLI-positive nuclei were seen in the
PVN, CeA, and the lPBN, as previously demonstrated with MTII
(40) and NDP-MSH (6; Fig.
5). c-Fos immunostaining of the lPBN by
-MSH may have arisen from activation of melanocortin-sensitive hypothalamic projections to the brain stem or by direct activation of
melanocortin receptors located in the lPBN by -MSH carried from the
third to the fourth ventricle. Because MTII delivered into either the
lateral or fourth ventricle is equally potent in suppressing food
intake and body weight (14), it is possible that -MSH
exerts direct effects in brain stem feeding areas that may explain
c-Fos induction in the lPBN.
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The parvocellular PVN, CeA, and lPBN are components of a pathway that
integrates centers regulating food intake and body weight with satiety
centers in the brain stem. The activation of all three areas by -MSH
suggests that it may enhance in the perception of satiety and thereby
facilitate the consumption of smaller meals, which, over time, results
in a modest decrease in body weight. However, meal size was not
measured in our study, and intracerebroventricularly administered
-MSH also increases mean arterial blood pressure via an action that
may involve the same brain nuclei (8). Further studies are
therefore warranted to differentiate activation by -MSH of
cardiovascular versus energy balance circuits along this pathway.
Perspectives
Previous studies have shown that leptin stimulates POMC expression in the Arc (7, 36), suggesting that melanocortin signaling may be downstream of leptin in a pathway leading to anorexia. Support for this hypothesis was provided by the observation that the anorectic properties of leptin can be blocked by pretreatment with the melanocortin receptor antagonist SHU-9119 (38) and that mice deficient in MC-4 receptor signaling have an attenuated response to leptin (25). Because the leptin receptor is expressed by Arc POMC neurons (7), these findings suggest that at least some of leptin's effects on energy homeostasis are mediated by increased-MSH signaling via the MC-4 receptor.
Our current findings, however, suggest that CNS leptin signaling must
involve pathways additional to melanocortin signaling, in agreement
with a previous study (4). For example, leptin's ability
to induce adipose-specific weight loss was not induced by chronic
intracerebroventricular -MSH, and the magnitude of -MSH-induced
anorexia and weight loss with chronic central administration is less
than that seen with leptin. In addition, CNS melanocortin signaling
must involve pathways not activated by leptin, because a pronounced
increase of cFLI-positive nuclei was seen in the lPBN in response to
intracerebroventricular -MSH, whereas intracerebroventricular leptin
does not have this effect (40).
These data support a model (Fig. 6) in
which -MSH induces reductions in food intake via activation of MC-4
receptors in both the brain stem and hypothalamus. On the basis of
previous studies, an increase of leptin delivery to the brain increases
forebrain MC-4-receptor signaling via both an increase in POMC
expression and a decrease in AgRP expression in Arc neurons. Increased
MC-4 signaling in second-order neurons involved in food intake
regulation may then promote anorexia. For example, activation of CRH-
or suppression of MCH-containing neurons may contribute to the response to MC-4-receptor stimulation, although we were unable to demonstrate significant effects of -MSH on either CRH or MCH mRNA levels. Thus
other neurons, such as thyrotropin-releasing hormone neurons of the PVN, may also contribute to the anorectic effects of -MSH (12).
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Leptin is hypothesized to affect food intake not only by stimulating
catabolic effector pathways such as the melanocortin system, but also
by inhibiting anabolic signaling systems such as NPY/AgRP neurons in
the Arc. As weight loss induced by chronic intracerebroventricular
-MSH proceeds, therefore, the decline in adiposity signaling (in the
form of circulating insulin and leptin) to the CNS is proposed to
activate NPY/AgRP neurons and thereby offset the anorectic action of
-MSH. This scenario is consistent with current literature and
provides a viable explanation for our results. If correct, the clinical
efficacy of melanocortin agonists in the treatment of obesity may be
limited by the effect of weight loss to activate compensatory responses
that antagonize the effect of melanocortins. This possibility suggests
that the efficacy of such compounds will be increased if used in
combination with drugs that block such compensatory responses, and
further studies are warranted to test this hypothesis.
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ACKNOWLEDGEMENTS |
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Many thanks to Dr. Todd Thiele for expert guidance and advice on the c-Fos assay and to Dr. George Merriam and Lynna C. Smith for the use of the DEXA scanner system located at the Veterans Affairs Puget Sound Health Care System, American Lake Division in Tacoma, WA. Technical assistance for in situ hybridization was provided by Vicki Hoagland and Will Anderson. The leptin assay was performed by Kimber Stanhope, the corticosterone assay by Elizabeth Colasurdo and Shannon Reed, the insulin assay by Hong Nguyen, and the glucose assay by Ruth Hollingworth. The riboprobe for POMC mRNA was a gift of Dr. Robert Steiner, and constructs for the MCH and AgRP riboprobes were provided by Dr. Tina M. Hahn.
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FOOTNOTES |
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This work was supported by grants from the National Institutes of Health (DK-12829, DK-52789, NS-32273, DK-50129, DK-35747, DK-17844, and DK-54080), the Diabetes Endocrinology Research Center, the Clinical Nutrition Research Unit and the Royalty Research Fund at the University of Washington, the American Diabetes Association, and the Veterans Affairs Merit Review Program.
Address for reprint requests and other correspondence: M. W. Schwartz, Dept. of Medicine, Univ. of Washington and Harborview Medical Center, Box 359757, 325 Ninth Ave., Seattle, WA 98104-2499 (E-mail: mschwart{at}u.washington.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 22 October 1999; accepted in final form 20 March 2000.
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TOP
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